Methods and compositions for rapid in vitro propagation of Swertia chirata

Abstract

The present disclosure relates to methods and compositions for in vitro cultivation of species of Swertia , e.g. Swertia chirata (Ham.). The disclosure provides culture media comprising Murashige and Skoog (MS) basal culture medium, plant hormones preferably selected from the group consisting of benzyladenine (BAP), gibberellic acid (GA 3 ), and auxins, and other additives, e.g. sucrose and agar. Preferably, auxins are selected from the group consisting of indole acetic acid (IAA), indole butyric acid (IBA), and naphthalene acetic acid (NAA). Individual plant hormone concentrations are preferably from about 0.5 mg/L to about 5.0 mg/L. The disclosure provides methods of in vitro cultivation of Swertia chirata comprising contacting preferably axillary bud and/or shoot apex explants with an initiation medium comprising a modified MS basal culture medium, BAP, IAA, IBA, and NAA to produce a primary explant, contacting the primary explant with a shoot propagation medium comprising, a modified MS basal culture medium, BAP, GA 3 , and IAA to produce a secondary explant, contacting a secondary explant with a rooting medium comprising a modified MS basal culture medium, IAA, IBA, and NAA. The methods and compositions of the invention are capable of inducing extraordinarily rapid in vitro propagation of Swertia chirata . The methods and compositions of the disclosure may be useful for conservation of this threatened species as well as producing bulk quantities, e.g. gram, kilogram or more, of plant material for medicinal

1. A culture medium comprising 1650 mg/L NH 4 NO 3 , 1900 mg/L KNO 3 , 440 mg/L CaCl 2 .2H 2 O, 370 mg/L MgSO 4 .7H 2 0, 170 mg/L KH 2 PO 4 , 0.83 mg/L KI, 6.2 mg/L H 3  BO 3 , 16.9 mg/L MnSO 4 .4H 2 0, 8.6 mg/L ZnSO 4 .7H 2 0, 0.025 mg/L Na 2  MoO 4 .2H 2 0, 0.025 mg/L CuSO 4 .5H 2 O, 0.025 mg/L CoCl 2 .6H 2 0, 0.5 mg/L nicotinic acid, 0.5 mg/L pyridoxine HCl, 0.1 mg/L thiamine, and 2 mg/L glycine, from about 0.5 mg/L to about 1.0 mg/L benzyladenine, from about 0.5 mg/L to about 1.0 mg/L IAA, from about 0.5 mg/L to about 1.0 mg/L IBA, about 0.5 mg/L NAA, and from about 1% to about 3% sucrose by weight and having a pH of about 5.8. 
     
     
       2. The culture medium of  claim 1  further comprising about 0.8% agar by weight. 
     
     
       3. A culture medium comprising 1650 mg/L NH 4 NO 3 , 1900 mg/L KNO 3 , 440 mg/L CaCl 2 .2H 2 O, 370 mg/L MgSO 4 .7H 2 0, 170 mg/L KH 2 PO 4 , 0.83 mg/L KI, 6.2 mg/L H 3 BO 3 , 16.9 mg/L MnSO 4 .4H 2 0, 8.6 mg/L ZnSO 4 .7H 2 0, 0.025 mg/L Na 2  MoO 4 .2H 2 0, 0.025 mg/L CuSO 4 .5H 2 O, 0.025 mg/L CoCl 2 .6H 2 0, 0.5 mg/L nicotinic acid, 0.5 mg/L pyridoxine HCl, 0.1 mg/L thiamine, and 2 mg/L glycine, from about 0.5 mg/L to about 1.0 mg/L BAP, about 1.0 mg/L GA 3 , about 1.0 mg/L IAA, and from about 1% to about 3% sucrose by weight and having a pH of about 5.8. 
     
     
       4. The culture medium of  claim 3  further comprising about 0.8% agar by weight. 
     
     
       5. A culture medium comprising 1650 mg/L NH 4 NO 3 , 1900 mg/L KNO 3 , 440 mg/L CaCl 2 .2H 2 O, 370 mg/L MgSO 4 .7H 2 0, 170 mg/L KH 2 PO 4 , 0.83 mg/L KI, 6.2 mg/L H 3  BO 3 , 16.9 mg/L MnSO 4 .4H 2 0, 8.6 mg/L ZnSO 4 .7H 2 0, 0.025 mg/L Na 2  MoO 4 .2H 2 0, 0.025 mg/L CuSO 4 .5H 2 O, 0.025 mg/L CoCl 2 .6H 2 0, 0.5 mg/L nicotinic acid, 0.5 mg/L pyridoxine HCl, 0.1 mg/L thiamine, and 2 mg/L glycine, from about 1.0 mg/L to about 5.0 mg/L IAA, from about 1.0 mg/L to about 5.0 mg/L IBA, from about 1.0 mg/L to about 5.0 mg/L NAA, and from about 1% to about 3% sucrose by weight and having a pH of about 5.8. 
     
     
       6. The culture medium of  claim 5  further comprising about 0.8% agar by weight.